Project Summary/Abstract Thrombosis is a leading cause of death in the United States. It is caused by excessive production of the blood clotting factor thrombin. During normal hemostasis, thrombin generation is tightly regulated by several anticoagulant proteins, including protein S (PS). PS is an important anticoagulant protein in humans with total deficiency producing severe, life-threatening thrombosis at birth. However, the biochemical mechanism(s) by which PS mediates its anticoagulant activity are poorly understood. PS is a cofactor for two other endogenous anticoagulants, tissue factor pathway inhibitor alpha (TFPIa) and activated protein C (APC). PS/TFPIa inhibits coagulation factor Xa (FXa), while PS/APC proteolytically inactivates factor Va (FVa). FVa and FXa combine to form prothrombinase, the powerful enzymatic complex that converts prothrombin to thrombin. Prothrombinase is resistant to inactivation by either PS/APC or PS/TFPIa during the propagation phase of blood clotting, yet its activity is clearly regulated because thrombi do not spread throughout the entire vasculature during normal hemostasis. PS promotes the inactivation of both protein components of prothrombinase, FVa (as an APC cofactor) and FXa (as a TFPIa cofactor), suggesting that it may modulate prothrombinase activity. We hypothesize that coordinated action of the PS/TFPIa and PS/APC anticoagulant systems will produce efficient inhibition of prothrombinase. This hypothesis will be tested via complimentary biochemical and structural studies. The K99 will focus on biochemical studies using assays established during my post-doctoral research. They will further define the mechanism by which PS enhances TFPIa inhibitory activity (Specific Aim 1.1) and determine how FXa inhibition by PS/TFPIa alters the ability of PS/APC to proteolytically inactivate FVa and vice versa (Specific Aim 1.2). The R00 will extend this work further by assessing the contribution of different physiologic membrane surfaces to the regulation of thrombin generation (Specific Aim 1.3). These functional studies will be correlated with structural studies. The K99 will focus on development of molecular models of the PS/TFPIa and PS/APC complexes (Specific Aim 2.1). The R00 will extend this work by producing crystal structures of individual domains of PS, TFPIa, and APC, separately and in complex (Specific Aims 2.2 and 2.3). These studies will coincide with career development training, which will include a focus on training in structural biology, through a combination of courses and work in the laboratory of Dr. Stephen Everse. This will differentiate my work from that of my mentor and enable me to establish an independent research career studying the biochemistry of hemostatic proteins.